REVIEW ARTICLE Open Access

Structural, functional, and evolutionaryanalysis of Cry toxins of Bacillusthuringiensis: an in silico studySujit Kumar Das1,2, Sukanta Kumar Pradhan1, Kailash Chandra Samal3 and Nihar Ranjan Singh4*

Abstract

Background: Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that synthesizes crystalline(Cry) protein, which is toxic and causing pathogenicity against mainly three insect orders: Coleoptera, Diptera, andLepidoptera. These crystalline protein inclusions, i.e., δ-endotoxins are successfully used as a bio-control agentagainst insect pests.

Main body: A total of 58 various Cry proteins belonging to these 3 insect orders were retrieved from SwissProtdatabase and are categorized into different groups. Structural and functional analysis were performed to understandthe functional domain arrangements at sequence level as well as at structural level involving both experimental andpredicted 3-dimensional models. Besides, the analysis of evolutionary relationship involving all 58 observed Cryproteins at the sequence, domain, and structural levels were done using different bioinformatics tools. Evolutionaryanalysis revealed that some Cry proteins having toxicity for a specific insect order are found to be clustered for anotherdifferent insect order, which concludes that they might have toxicity for more than one insect order. Three-dimensional (3D) structure analysis of both experimental and predicted models revealed that proteins might havetoxicity for a specific insect order differ in their structural arrangements and was observed in Cry proteins belonging to3 different insect orders.

Conclusions: It could be hypothesized that an inner-molecular domain shift or domain insertion/deletion might havetaken place during the evolutionary process, which consequently causes structural and functional divergence of Bt. Thestudy output may be helpful for understanding the diversity as well as specificity of the analyzed insecticidal proteinsand their application as a biopesticide in the field of agriculture.

Keywords: Bacillus thuringiensis, Biopesticides, Cry toxins, Bioinformatics, Phylogenetic analysis

BackgroundBacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium causing pathogenicity in insects. Btstrains synthesize Crystal (Cry) and cytolytic (Cyt) toxins(also known as δ-endotoxins) that have a natural insecti-cidal effect on selective insect orders. δ-endotoxins of Btare being used successfully as a biological control agentagainst some insect pests (Schnepf 1995). These

endotoxins are exclusively active against larval stages ofdifferent insect orders such as Lepidoptera (Butterflies,Moths), Coleoptera (Flies and Mosquitoes), and Diptera(Beetles and Weevils) (Raymond et al. 2010). Duringsporulation phase, Bt produces these Cry or Cyt toxinsthat have hazardous effect on insects (Bravo et al. 2011).Cry and Cyt toxins are considered as parasporal inclusionproteins from Bt that exhibit toxic effects and hemolyticactivity respectively (Crickmore et al. 1998).These twotypes of toxins belong to a class of pore-forming toxins(PFTs) that are secreted as water-soluble proteins andundergo conformational changes in order to insert into

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* Correspondence: nihar.singh@gmail.com4Department of Botany and Center of Excellence in Environment and PublicHealth, Ravenshaw University, Cuttack, Odisha 753003, IndiaFull list of author information is available at the end of the article

Egyptian Journal ofBiological Pest Control

Das et al. Egyptian Journal of Biological Pest Control (2021) 31:44 https://doi.org/10.1186/s41938-021-00394-6

the host membrane. When Crystal (Cry) toxins ingestedby insects are get solubilized in their midgut, then it getsproteolytically activated by midgut proteases and bind tospecific receptors located in the insect cell membraneleading to cell disruption and cell death (Bravo et al.2007). Most Cry proteins exist as inactive protoxins thatcan be converted into active toxins by certain kinds of in-sect midgut proteases (Höfte and Whiteley 1989). This ac-tivation process appears to involve a sequential series ofproteolytic cleavages, starting at the C-terminus and pro-ceeding toward the N-terminus until the protease-stabletoxin is generated (Choma and Kaplan 1990). Besidesthese membrane proteins, other components have beenidentified due to their capacity to interact with 3d-Crytoxins (3 domain-Cry toxins) such as glycolipids, intracel-lular proteins, V-ATPase subunit A or actin (McNall andAdang 2003; Griffitts et al. 2005; Bayyareddy et al. 2009).The interaction of Cry1 toxins with different proteinspresent in lepidopteran midgut cells is a complex processinvolving multiple membrane proteins such as cadherin-like proteins (CADs), aminopeptidase N (APN), and alka-line phosphatase (ALP) (Pigott and Ellar 2007; Soberonet al. 2009).Extensive screening of Bt strains and Cry gene sequen-

cing has led to the identification of more than 700 Crygene sequences (Höfte and Whiteley 1989; Van Franken-huyzen 2009; Crickmore et al. 2011). These sequenceshave been classified according to their amino acid se-quence identities in 70 different Cry gene groups (Cry1,Cry2...Cry 70) where toxins belonging to each groupshare less than 40% amino acid identity with proteinsfrom other groups (Crickmore et al. 1998). Within eachgroup, a capital letter (Cry1A, Cry1B, etc.) is given whenthey share less than 70% identity. A small letter (Cry1Aa,Cry1Ab, etc.) is given when toxins share more than 70%but less than 95% identity (Bravo et al. 2013). Many Cryproteins show pesticidal properties, while some proteinproduced as parasporal crystals that have unknown in-vertebrate targets called as parasporins such as Cry31A,Cry41A, Cry45A, Cry46A, Cry63A, and Cr64A exhibitstrong and specific cytocidal activity against human can-cer cell (Mizuki et al. 2000). Phylogenetic analysis of Crytoxins shows that the great variability in the biocidal ac-tivity of the 3d-Cry group has resulted from the inde-pendent evolution of the 3 structural domains and thedomain III swapping among different toxins. These twoprocesses have generated proteins with similar modes ofaction but with different specificities (de Maagd et al.2001). Various 3-dimensional crystal structures of acti-vated Cry toxins have been determined by experimentalmethods. These are Coleoptera-specific Cry3Aa (PDBentry: 1DLC) (Li et al. 1991) and Cry3Bb1 (PDB entry:1JI6) (Galitsky et al. 2001), Cry 34 Ab1(PDB entry:4JOX) and Cry 35 Ab1 (PDB entry: 4JPo) (Kelker et al.

2014), Lepidoptera-specific Cry1Aa (PDB entry: 1CIY)(Grochulski et al. 1995), Cry1Ac (PDB entry: 4ARX/4ARY/4W8J) (Evdokimov et al. 2014), Cry1Da (PDBentry: 6OVB) (Wang et al. 2019), Cry1Fa (PDB entry:6DJ4) (Wang et al. 2018), Cry1Be (PDB entry: 6OWK)(Wang et al. 2019), Lepidoptera/Diptera-specific Cry2Aa(PDB entry: 1I5P) (Morse et al. 2001), Diptera-specificCry4-Ba (PDB entry: 1W99) (Boonserm et al. 2005), andCry4Aa (PDB entry: 2C9K) (Boonserm et al. 2006).Although these toxins exhibit markedly different insecti-cidal specificities, the overall folding patterns of theirstructures are quite similar, comprising 3 domains. Thedomain (I) is a bundle of 7–8 α helices involved in poreformation, domain (II) is a β-prism with exposed loopsregions involved in receptor binding, and domain (III) isa β-sandwich and has influence on receptor binding, ionchannel formation, and insect specificity (Li et al. 1991;Grochulski et al. 1995; Derbyshire et al. 2001; Galitskyet al. 2001; Morse et al. 2001; Guo et al. 2009, and Huiet al. 2012).It has been observed that the Crystal toxins showed spe-

cificity to different insect orders which signify that theymight have shared certain level of relationships at differentlevels such as sequence, domain, and structure. Thepresent article aimed to retrieve different Cry proteinsfrom various protein databases, followed by phylogeneticanalysis, domain characterization, structure predictions,experimental structure analysis, and comparison.

Main textSequence retrieval and analysis of Cry proteinsCrystal (Cry) proteins of Bt having specificity to 3 broadinsect orders: Lepidoptera, Coleoptera, and Diptera wereretrieved from the UniprotKB protein sequence database(https://www.uniprot.org/). Selections were made onlyfrom manually annotated and reviewed sequences be-longing to Swiss-Prot and stored in FASTA format forfurther bioinformatics analysis.

Domain identification, characterization, and functionalanalysisIdentification and annotation of genetically mobile do-mains and its architectures in the manually annotatedand reviewed protein sequences were carried out using aweb-based bioinformatics domain prediction tool, i.e.,NCBI Batch CD-Search service (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). These domainsare extensively annotated with respect to phyletic distri-butions, functional class, tertiary structures, and func-tionally important residues.

Sequence alignment and phylogenetic tree constructionDivergence analysis among the retrieved Crystal proteinsand the predicted domains were conducted in Molecular

Das et al. Egyptian Journal of Biological Pest Control (2021) 31:44 Page 2 of 14

Evolutionary Genetics Analysis X (MEGA X) (Kumaret al. 2018) using Neighbor-Joining (NJ) method (Saitouand Nei 1987). The predicted phylogenetic tree was eval-uated using bootstrap reliability test for 1000 replicates.The tree was drawn to scale, with branch lengths in thesame units as those of the evolutionary distances used toinfer the phylogenetic tree. The evolutionary distanceswere computed using the Poisson correction method(Zuckerkandl and Pauling 1965) and are in the units ofthe number of amino acid substitutions per site.

Structural diversity analysis and predictionThe selected Cry proteins belonging to the 3 major insectorders Lepidoptera, Coleoptera, and Diptera weresearched in RCSB-PDB (Research Collaborator for Struc-tural Bioinformatics-Protein Data Bank) (https://www.rcsb.org/) for availability of experimentally solved 3-dimensional structures and PDB files were downloaded.

The protein sequences having no 3-dimensional struc-tures in PDB were submitted to web based Protein Hom-ology/analogY Recognition Engine V2.0 (Phyre2) (http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) forprotein modeling and prediction of 3-dimensionalstructures.

Crystal (Cry) proteins of Bacillus thuringiensisTotal 58 Cry protein sequences of bacteria Bt were re-trieved from UniProtKB database (only annotated andreviewed). Among all of these 58 sequences, total 15, 10,and 33 numbers of Cry proteins were observed in the in-sect orders: Coleoptera, Diptera, and Lepidoptera, re-spectively. Primary sequence variation in length of Cryproteins were noticed as 123–1169 amino acids in caseof order Coleoptera (Table 1), 643–1180 amino acids inDiptera (Table 2), and mostly more than 1100 aminoacids in Lepidoptera (Table 3).

Table 1 List of cry proteins having toxicity for Coleoptera insect order

Entry Protein Gene Organism Length PDB ID Insect_Order

P17969 cry3Ba cry3Ba Bacillus thuringiensis subsp. tolworthi 659 N/A Coleoptera

P0A379 cry3Aa cry3Aa Bacillus thuringiensis subsp. tenebrionis 644 1DLC,4QX0,4QX1,4QX2,4QX3 Coleoptera

P0A381 cry3Aa cry3Aa Bacillus thuringiensis subsp. san diego 644 N/A Coleoptera

P0A380 cry3Aa cry3Aa Bacillus thuringiensis subsp. morrisoni 644 N/A Coleoptera

Q45744 cry3Ca cry3Ca Bacillus thuringiensis subsp. kurstaki 649 N/A Coleoptera

Q45704 cry8Aa cry8Aa Bacillus thuringiensis subsp. kumamotoensis 1157 N/A Coleoptera

Q45705 cry8Ba cry8Ba Bacillus thuringiensis subsp. kumamotoensis 1169 N/A Coleoptera

Q45708 cry7Ab cry7Ab Bacillus thuringiensis subsp. kumamotoensis 1138 N/A Coleoptera

Q45706 cry8Ca cry8Ca Bacillus thuringiensis subsp. japonensis 1160 N/A Coleoptera

O06014 cry9Da cry9Da Bacillus thuringiensis subsp. japonensis 1169 N/A Coleoptera

Q45707 cry7Ab cry7Ab Bacillus thuringiensis subsp. dakota 1138 N/A Coleoptera

Q939T0 cry34Ab1 N/A Bacillus thuringiensis 123 4JOX Coleoptera

Q03749 cry7Aa cry7Aa Bacillus thuringiensis 1138 N/A Coleoptera

Q939S9 cry35Ab1 N/A Bacillus thuringiensis 383 4JP0 Coleoptera

Q06117 cry3Bb1 cry3Bb1 Bacillus thuringiensis 652 1JI6 Coleoptera

Table 2 List of cry proteins having toxicity for Diptera insect order

Entry Protein Gene Organism Length PDB ID Insect_Order

Q9ZIU5 cry11Bb cry11Bb Bacillus thuringiensis subsp. medellin 750 N/A Diptera

O32307 cry19Aa cry19Aa Bacillus thuringiensis subsp. Jegathesan 648 N/A Diptera

Q45730 cry11Ba cry11Ba Bacillus thuringiensis subsp. Jegathesan 724 N/A Diptera

P05519 cry4Ba cry4Ba Bacillus thuringiensis subsp. Israelensis 1136 1W99,4MOA Diptera

P09662 cry10Aa cry10Aa Bacillus thuringiensis subsp. Israelensis 675 N/A Diptera

P16480 cry4Aa cry4Aa Bacillus thuringiensis subsp. Israelensis 1180 2C9K Diptera

P21256 cry11Aa cry11Aa Bacillus thuringiensis subsp. Israelensis 643 N/A Diptera

Q9S597 cry27Aa cry27Aa Bacillus thuringiensis subsp. higo 826 N/A Diptera

O86170 cry19Ba cry19Ba Bacillus thuringiensis subsp. higo 682 N/A Diptera

O32321 cry20Aa cry20Aa Bacillus thuringiensis subsp. fukuokaensis 753 N/A Diptera

Das et al. Egyptian Journal of Biological Pest Control (2021) 31:44 Page 3 of 14

Analysis of Cry proteins structural domainsPrimary sequences of 58 Cry proteins with different spe-cificity in the 3 insect orders were further analyzed toannotate different structural domains involved in variousbiological processes (Tables 4, 5, and 6). Domain analysiswas revealed that most of the Cry proteins have 3 struc-tural domains such as Endotoxin_N (PF03944), Endo-toxin_M (PF00555), and Endotoxin_C (PF03945) (Tables4, 5, and 6). It was also observed that Endotoxin_N,Endotoxin_M, and Endotoxins_C are presented in Nterminus, middle, and C-terminal region of the protein

(Figs. 1 and 2), respectively. Generally, N-terminal hel-ical domain involves in membrane insertion and poreformation whereas middle and C-terminal domains havea vital role in receptor bindings.

Evolutionary analysis of Cry proteins and domainsEvolutionary tree was constructed for 58 Cry proteinsusing MEGA X tool after elimination of all residual posi-tions containing gaps and was further analyzed (Fig. 3).The tree is clustered into 3 major groups such as clustersI, II, and III. Cluster I (red color) consists of Cry proteins

Table 3 List of cry proteins having toxicity for Lepidoptera insect order

Entry Protein Gene Organism Length PDB ID Insect_Order

Q9ZAZ5 cry1Bd cry1Bd Bacillus thuringiensis subsp. wuhanensis 1231 N/A lepidoptera

Q9ZAZ6 cry1Gb cry1Gb Bacillus thuringiensis subsp. wuhanensis 1169 N/A lepidoptera

Q45733 cry9Ca cry9Ca Bacillus thuringiensis subsp. tolworthi 1157 N/A lepidoptera

Q45729 cry15Aa cry15Aa Bacillus thuringiensis subsp. thompsoni 340 N/A lepidoptera

P0A369 cry1Aa cry1Aa Bacillus thuringiensis subsp. sotto 934 N/A lepidoptera

Q45715 cry1Ka cry1Ka Bacillus thuringiensis subsp. morrisoni 1215 N/A lepidoptera

Q45718 cry1Hb cry1Hb Bacillus thuringiensis subsp. morrisoni 1155 N/A lepidoptera

O66377 cry1Fb cry1Fb Bacillus thuringiensis subsp. morrisoni 1169 N/A lepidoptera

P0A370 cry1Ab cry1Ab Bacillus thuringiensis subsp. kurstaki 1155 N/A lepidoptera

P0A366 cry1Aa cry1Aa Bacillus thuringiensis subsp. kurstaki 1176 1CIY lepidoptera

P21254 cry2Ab cry2Ab Bacillus thuringiensis subsp. kurstaki 633 N/A lepidoptera

P05068 cry1Ac cry1Ac Bacillus thuringiensis subsp. kurstaki 1178 4ARX, 4ARY,4W8J lepidoptera

Q57458 cry1Ea cry1Ea Bacillus thuringiensis subsp. kenyae 1171 N/A lepidoptera

Q99031 cry9Aa cry9Aa Bacillus thuringiensis subsp. galleriae 1156 N/A lepidoptera

P0A368 cry1Aa cry1Aa Bacillus thuringiensis subsp. entomocidus 1176 N/A lepidoptera

P0A375 cry1Ca cry1Ca Bacillus thuringiensis subsp. entomocidus 1189 N/A lepidoptera

Q03748 cry1Ae cry1Ae Bacillus thuringiensis subsp. alesti 1181 N/A lepidoptera

P0A376 cry1Ca cry1Ca Bacillus thuringiensis subsp. aizawai 1189 N/A lepidoptera

P0A367 cry1Aa cry1Aa Bacillus thuringiensis subsp. aizawai 1176 N/A lepidoptera

Q03744 cry1Ad cry1Ad Bacillus thuringiensis subsp. aizawai 1179 N/A lepidoptera

P19415 cry1Da cry1Da Bacillus thuringiensis subsp. aizawai 1165 6OVB lepidoptera

Q03745 cry1Eb cry1Eb Bacillus thuringiensis subsp. aizawai 1174 N/A lepidoptera

Q03746 cry1Fa cry1Fa Bacillus thuringiensis subsp. aizawai 1174 6DJ4 lepidoptera

Q9ZNL9 cry9Ea cry9Ea Bacillus thuringiensis subsp. aizawai 1150 N/A lepidoptera

Q45746 cry1Ga cry1Ga Bacillus thuringiensis 1166 N/A Lepidoptera

Q9XDL1 cry1Id cry1Id Bacillus thuringiensis 719 N/A Lepidoptera

O85805 cry1Be cry1Be Bacillus thuringiensis 1227 6OWK Lepidoptera

Q45748 cry1Ha cry1Ha Bacillus thuringiensis 1172 N/A Lepidoptera

Q45716 cry1Jb cry1Jb Bacillus thuringiensis 1170 N/A Lepidoptera

Q9S515 cry1Ag cry1Ag Bacillus thuringiensis 1176 N/A Lepidoptera

Q45747 cry1Db cry1Db Bacillus thuringiensis 1160 N/A Lepidoptera

Q45738 cry1Ja cry1Ja Bacillus thuringiensis 1167 N/A Lepidoptera

Q45739 cry1Bb cry1Bb Bacillus thuringiensis 1229 N/A Lepidoptera

Das et al. Egyptian Journal of Biological Pest Control (2021) 31:44 Page 4 of 14

having toxicity for insect order Lepidoptera. Interestingly,all Cry1A, Cry1D, Cry1E, Cry1H, Cry1F, Cry1G, Cry1C,and Cry1J are fallen in this cluster. However, in cluster II(green color), all Cry proteins show toxicity towards Cole-optera are joined together along with Cry11D, Cry9Ca,Cry1Be, Cry1Ka, Cry1Bd, Cry1Bb, and Cry9Ea. But,Cry9Aa and Cry2Ab targets for Lepidoptera and Cry8Catargets Coleoptera were obtained as a member of clusterIII (blue color) which shows toxicity towards Diptera.Interestingly, lepidopteran target Cry15Aa and coleop-teran targets Cry34Ab1 and Cry35Ab1 were seemed as an

out-group in the whole tree and were found to be distantlyrelated from all the Cry protein sequences. The Cry pro-teins for the specific order were found to be diverged fromthe own group and placed in different insect orders indi-cate that they might have insecticidal property for morethan one insect order. Besides, in order to understand theinsect specificity of these Cry proteins, the functional do-main regions were also taken for studying the divergenceamong them. The phylogenetic tree of the domain Endo-toxin_N, Endotoxin_M, and Endotoxin_C was con-structed using MEGA X software and presented (Fig. 4).

Table 4 List of cry protein domains having toxicity for Coleoptera insect order

Protein name UniprotaccessionNo

Sequencelength

Endotoxin_N E value Endotoxin_M E value Endotoxin_C E value

Start End Start End Start End

cry3Ba_coleoptera P17969 659 73 296 4.4e−30 304 510 9.6e−62 520 659 2.5e−41

cry3Aa_coleoptera_1 P0A379 644 65 287 3.3e−30 295 499 2.1e−62 509 644 7.6e−41

cry3Aa_coleoptera_2 P0A381 644 65 287 3.3e−30 295 499 2.1e−62 509 644 7.6e−41

cry3Aa_coleoptera_3 P0A380 644 65 287 3.3e−30 295 499 2.1e−62 509 644 7.6e−41

cry3Ca_coleoptera Q45744 649 64 285 2.9e−32 293 502 3.6e−58 512 649 1e−39

cry8Aa_coleoptera Q45704 1157 69 289 1.1e−27 297 516 2.6e−44 526 662 6.8e−46

cry8Ba_coleoptera Q45705 1169 80 290 8.8e−39 298 512 7.3e−54 522 658 9.3e−36

cry7Ab_coleoptera_1 Q45708 1138 61 278 1.8e−29 286 487 9.7e−59 497 637 1.5e−42

cry8Ca_coleoptera Q45706 1160 77 290 3.5e−26 298 503 5.7e−50 513 659 2.6e−33

cry9Da_coleoptera O06014 1169 73 293 5.6e−26 301 521 1.7e−57 531 668 2.7e−48

cry7Ab_coleoptera_2 Q45707 1138 61 278 2.6e−30 286 487 2.5e−57 497 637 3.7e−41

cry34Ab1_coleopteraa Q939T0 123 – – – – – – – – –

cry7Aa_coleoptera Q03749 1138 63 278 1.5e-27 286 487 2e-59 497 637 1.9e-42

cry35Ab1_coleopterab Q939S9 383 – – – – – – – – –

cry3Bb1_coleoptera Q06117 652 65 288 1.2e−29 296 502 1.6e−63 512 651 1e−42

Toxin_10: domain position (174–348)aAegerolysin: domain position (3–118)bRICIN: domain position (4–138)

Table 5 List of cry protein domains having toxicity for Diptera insect order

Protein name Uniprotaccessionno.

Sequencelength

Endotoxin_N E value Endotoxin_M E value Endotoxin_C E value

Start End Start End Start End

cry11Bb_diptera Q9ZIU5 750 56 240 2.7e−21 – – – – – –

cry19Aa_diptera O32307 648 69 292 4.3e−25 300 502 3.6e−49 512 648 1.2e−31

cry11Ba_diptera Q45730 724 55 240 1.2e−18 – – – – – –

cry4Ba_diptera P05519 1136 52 268 1.1e−26 283 470 2.1e−40 480 634 2.2e−40

cry10Aa_diptera P09662 675 95 300 2.9e−23 308 500 5.9e-37 510 650 2.8e−35

cry4Aa_diptera P16480 1180 80 314 1.3e-21 322 528 2.6e-54 538 678 2.6e-40

cry11Aa_diptera P21256 643 41 240 5.1e−23 – – – – – –

cry27Aa_diptera Q9S597 826 92 302 6.4e−23 311 499 5.1e−19 524 683 8.5e−34

cry19Ba_diptera O86170 682 71 280 3.2e−32 293 496 2.2e−48 506 638 9.1e−34

cry20Aa_diptera O32321 753 67 280 1.7e−22 288 485 3.2e−44 495 631 1.5e−26

Das et al. Egyptian Journal of Biological Pest Control (2021) 31:44 Page 5 of 14

The Cry protein sequence Endotoxin_N domains (55numbers) was clustered into 3 major groups. The evolu-tionary analysis (Fig. 4a) depicted that the divergencepattern of all the domain sequences as similar with thatof all Cry protein sequences and presented in (Fig. 3).Evolutionary analysis of Endotoxin_M domains (51numbers) present in Crystal protein target for all the 3insect orders depicted in (Fig. 4b) showed a similar typeof divergence in the phylogenetic tree as that of two pre-dicted tree for Endotoxin_N and total Cry protein

sequence. Phylogenetic tree involving Endotoxin_C do-mains (51 numbers) was revealed that all Cry1 and Cry9targets for lepidopterans are grouped into a single clus-ter, except for Cry1C, Cry1B, Cry1E, and Cry1Ac, whichare found to be clustered into the second group ofCry protein, which targets the coleopterans. However,all Cry proteins for dipteral were clustered into thesame group, except for Cry8Ba, which targets thecoleopteran (Fig. 4c).

Table 6 List of protein domains having toxicity for Lepidoptera insect order

Protein name Uniprotaccessionno.

Sequencelength

Endotoxin_N E value Endotoxin_M E value Endotoxin_C E value

Start End Start End Start End

cry1Bd_lepidoptera Q9ZAZ5 1231 59 275 2.4e−26 283 495 2.1e−65 505 643 1.1e−38

cry1Gb_lepidoptera Q9ZAZ6 1169 45 245 4.7e−22 253 449 4.8e−54 459 596 3.7e−48

cry9Ca_lepidoptera Q45733 1157 70 290 1.6e−29 298 505 3.8e-56 515 658 6e−41

cry15Aa_lepidopteraa Q45729 340 – – – – – – – – –

cry1Aa_lepidoptera_1 P0A369 934 46 251 1e−31 259 460 4.9e−53 470 607 8.9e−47

cry1Ka_lepidoptera Q45715 1215 56 276 2.7e−25 284 490 6.1e−60 500 637 4.8e−40

cry1Hb_lepidoptera Q45718 1155 48 248 3.6e−28 256 454 2.3e−58 464 596 3.2e−38

cry1Fb_lepidoptera O66377 1169 44 249 5.2e−33 257 454 6.2e−54 464 600 1.6e−49

cry1Ab_lepidoptera P0A370 1155 46 251 8.2e−31 259 461 1.6e−57 471 608 2.6e−46

cry1Aa_lepidoptera_2 P0A366 1176 46 251 2.3e−31 259 460 1e−52 470 607 2.6e−46

cry2Ab_lepidoptera P21254 633 53 263 2.5e−31 – – – – – –

cry1Ac_lepidoptera P05068 1178 46 251 3.5e−31 259 461 3.6e−58 471 609 1.9e−36

cry1Ea_lepidoptera Q57458 1171 44 250 3e−28 258 454 1.6e−60 464 601 2.2e−39

cry9Aa_lepidoptera Q99031 1156 61 287 5.7e−17 295 510 5.3e−46 520 656 1.4e−36

cry1Aa_lepidoptera_3 P0A368 1176 46 251 2.3e−31 259 460 1e−52 470 607 2.6e-46

cry1Ca_lepidoptera_1 P0A375 1189 42 250 9.1e−28 260 457 1.4e−51 467 616 1.5e−37

cry1Ae_lepidoptera Q03748 1181 46 251 5.5e−29 259 461 3.7e−57 471 608 2.8e−46

cry1Ca_lepidoptera_2 P0A376 1189 42 250 6.2e−28 260 457 5.1e−54 467 616 1.5e−37

cry1Aa_lepidoptera_4 P0A367 1176 46 251 3.4e−31 259 460 1e−52 470 607 2.6e−46

cry1Ad_lepidoptera Q03744 1179 46 251 9.6e−31 259 460 5.8e−53 470 607 1.2e−44

cry1Da_lepidoptera P19415 1165 45 250 1.9e−32 258 450 1.4e−54 460 592 7e−39

cry1Eb_lepidoptera Q03745 1174 42 249 8.5e−31 257 453 2e−59 463 599 1.4e−39

cry1Fa_lepidoptera Q03746 1174 44 249 8.7e−33 257 454 1.4e−53 464 601 2.2e−43

cry9Ea_lepidoptera Q9ZNL9 1150 71 293 3.9e−22 301 505 2.6e−47 515 651 2.1e−42

cry1Ga_lepidoptera Q45746 1166 46 245 1.7e−26 253 446 9.4e−51 456 593 2.9e−49

cry1Id_lepidoptera Q9XDL1 719 64 279 1.8e−33 287 497 6e−65 507 644 2.3e−48

cry1Be_lepidoptera O85805 1227 60 275 5.8e−25 283 493 6.9e−65 503 639 5.8e−39

cry1Ha_lepidoptera Q45748 1172 48 249 8.2e−28 257 455 8.3e−58 465 599 5.6e−35

cry1Jb_lepidoptera Q45716 1170 46 250 6.9e−27 258 449 8.6e−57 459 596 1.1e−43

cry1Ag_lepidoptera Q9S515 1176 46 251 2.9e−31 259 454 2e−44 473 607 1.6e−37

cry1Db_lepidoptera Q45747 1160 45 250 4e−31 258 450 4e−54 460 592 1.7e−38

cry1Ja_lepidoptera Q45738 1167 46 250 6.8e−27 258 449 1.7e−54 459 595 8.1e−49

cry1Bb_lepidoptera Q45739 1229 60 275 2.4e−26 283 495 2.1e−65 505 641 6.3e−46aETX_MTX2: domain position (40–265)

Das et al. Egyptian Journal of Biological Pest Control (2021) 31:44 Page 6 of 14

Analysis of Cry protein structureStructural analysis of Cry protein sequences was per-formed to understand its function in a better way. It hasbeen observed that most of the Cry proteins do not haveexperimentally solved 3-dimensional structures in theRCSB PDB, except for Cry3Aa, Cry34Ab1, Cry35Ab1,and Cry3Bb1 for Coleoptera (Fig. 5a), Cry4Ba andCry4Aa for Diptera (Fig. 5b), and Cry1Aa, Cry1Ac,Cry1Da, Cry1Fa, and Cry1Be for Lepidoptera (Fig. 6a,b).The rest 47 unsolved 3-dimensional structures of Cryproteins belongs to 3 different insect orders predicted

through Phyre2 homology modeling server, i.e., 11 num-bers of model structure for coleopteran, 08 numbers ofmodel structures for dipterans, and 28 numbers ofmodel structure for lepidopterans. Structural analysis ofCry proteins for coleopteran’s group revealed that theproteins having Endotoxin_N, Endotoxin_M, and Endo-toxin_C at sequence level corresponding to domain I,domain II, and domain III, respectively, at their struc-tural levels. Domain I regions found to be observedstarting from 60 to 300 amino acids at the N-terminalregion of the Cry proteins and consists of α-helices (~8

Fig. 1 a NCBI batch conserved domain-search result for Coleoptera insect order. b NCBI batch conserved domain-search for Diptera insect order

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numbers) whereas amino acid position 280–510 formdomain II which consists of ant parallel β-sheets andshort helices. Domain III is a β-sandwich of two ant par-allel highly twisted β-sheets and comprises 490–670

amino acid residues, corresponding to Endotoxin_C do-main of the Cry protein at the C-terminal region. Bydoing structure-structure alignment involving both ex-perimental and structural Cry proteins of Coleopteran

Fig. 2 NCBI batch conserved domain-search for Lepidoptera insect order

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group depicted that all 15 three-dimensional structuresfor Coleopteran are clustered into two major groupscomprising Cry7Aa, Cry9Da, Cry8Ba, Cry8Ca, Cry7Ab(2 numbers), and Cry8Aa in one group whereas Cry3Aa(3 numbers), Cry3Ca, Cry3Ba, and Cry3 in anothergroup whereas Cry34Ab1 and Cry35Ab1 are observed asout-groups (Fig. 7a). The divergence level may be due tothe absence of Endotoxin_N, Endotoxin_M, and Endo-toxin_C domains. In case of Cry proteins having specifi-city for Diptera, 8 model structures were predicted suchas Cry10Aa, Cry11Aa, Cry11Ba, Cry11Bb, Cry19Aa,

Cry19Ba, Cry20Aa, and Cry27Aa. Three-dimensionalstructural analysis of both experimental (2 numbers) andmodel (8 numbers) structure revealed that domain I ispresent in all the protein which extends from residue40–302 is totally α-helical and contains around 8 num-bers of α-helices whereas domain II and domain III spanover residue 280–530 and 480–685, respectively, exceptCry11Ba and Cry11Aa whereas Endotoxin_M and Endo-toxin_C domains are absent. In structural neighbor ana-lysis, all are grouped in single group except Cry4Bawhich is found to be an out-group for this group (Fig.

Fig. 3 Phylogenetic tree using MEGA X showing 58 numbers of cry proteins having toxicity for three insect orders

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7b). A total of 28 numbers of predicted protein struc-tures having specificity for lepidopteran’s group werepredicted. It was revealed that Cry1Aa, Cry1Be, Cry1Ba,and Cry1fa have the same type of folding patternwhereas totally different orientation was observed in caseof Cry15Aa due to absence of Endotoxin_N, Endotoxin_M, and Endotoxin_C domains. Different domain regionssuch as domain I, domain II, and domain III extendfrom 40 to 295, 253–510, and 458–660, respectively, forlepidopteran’s group (Table 6). Alignment of all 28 pro-teins structures was clustered into one major group leav-ing behind Cry2Ab as distantly related to them whereasCry15Aa was observed as out-group (Fig. 7c).B. thuringiensis (Bt) strains produce a wide variety of

proteins having toxicity against diverse insect orders.These toxins classified into 2 major groups: crystal (Cry)and cytolytic (Cyt). More than 700 Cry gene sequencesthat code for crystal protein (Cry) have been identifiedin plasmids by several researchers (Höfte and Whiteley1989; Schnepf et al. 1998; Van Frankenhuyzen 2009).

Many Cry proteins are reported to have useful insecti-cidal properties for controlling insect pests in agriculture(Sanchis and Bourguet 2008). However, strong cytocidalactivities have also been noticed against vertebrates(Palma et al. 2014). Primary protein sequence databasesearch revealed that there are 58 numbers of Cry pro-teins showing specificity towards the 3 major insect or-ders: Coleoptera, Diptera, and Lepidoptera (Donovanet al. 2016; Sanchis and Bourguet 2008; Naimov et al.2008). Sequences of different insect orders showed dif-ferent sequence’s length. All Cry1, Cry2, and Cry15groups showed toxicity towards lepidopteran insects,whereas Cry9 group showed toxicity towards Lepidop-tera and Coleoptera. Similarly, Cry proteins such asCry3, Cry7, Cry8, Cry34, and Cry35 groups were foundto have specificity for the coleopterans. Cry4, Cry10,Cry11, Cry19, Cry20, and Cry27 groups exhibit insecti-cidal activity against the insect belonging to dipteran’sorder. The classification of Cry proteins and their in-secticidal activity against specific insect orders have been

Fig. 4 a Phylogenetic tree of Cry protein domains for Coleoptera using MEGA X. b Phylogenetic tree of Cry protein domains for Diptera usingMEGA X. c Phylogenetic tree of Cry protein domains for Lepidoptera using MEGA X

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Fig. 5 a Experimental and predicted 3D structures (model) of Cry proteins having toxicity for Coleoptera insects. b Experimental and predicted3D structures (model) of Cry proteins having toxicity for Diptera insects

Fig. 6 a, b Experimental and predicted 3D structures (model) of Cry proteins having toxicity for Lepidoptera insects

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studied by several researchers (Crickmore 2000; deMaagd et al. 2001). Analysis of evolutionary relationshiprevealed that Cry proteins such as Cry1Id, Cry9Ca,Cry1Be, Cry1Ka, Cry1Bd, Cry1Bb, and Cry9Ea showedtoxicity towards lepidopteran’s insects also clustered to-gether with the Cry proteins having toxicity for the cole-opterans. This finding is supported by (Crickmore 2000).It has been also suggested that these proteins may havetoxicity against both insect orders and was later showedfor Cry1B toxin (López-Pazos et al. 2009). Similarly,Cry8Ca proteins of coleopteran’s group and Cry2Ab andCry9Aa for lepidopterans are found to be clustered inDiptera insect group. This phylogenetic relationship ofwhole Cry proteins could not able to reveal how Crytoxin involves in insect specificity. To validate further,phylogenetic analysis of the 3 structural domains such asdomain I (Endotoxin_N), domain II (Endotoxin_M), anddomain III (Endotoxin_C) were carried out independ-ently. Divergence pattern were observed similar in caseof both sequence level and structural level with minorfluctuation.As per literature, the domain swapping of different Cry

toxin is likely to be an active evolutionary process for de-termining insect specificity (de Maagd et al. 2001). Three-dimensional X-ray crystallography structures of severalCry toxins of Bt have been reported in this connection. Atotal of 11 numbers of three-dimensional structures of theCry proteins were retrieved from RCSB PDB out of which

4 proteins for Coleoptera, 2 for Diptera, and 5 for Lepi-doptera. Homology model structure for 47 Cry proteinswere predicted using web based Phyre2 tool to study thestructural arrangements of 3 different domains. All experi-mental and predicted models of Cry proteins revealed thatthe domain I is present in N-terminal region having α-helices, domain II consists of three antiparallel β-sheets,and domain III consists of two twisted anti-parallel β-sheets forming a sandwich. This observations and findingsare also supported by different published reports (Gro-chulski et al. 1995; de Maagd et al. 2001, and Gouet et al.2003). Structure-structure alignment was also carried outinvolving all the 58 numbers of Cry proteins (experimentaland model 3-dimensional structures) in order to under-stand the divergence among each other. In case of Coleop-tera insect order, 3-dimensional structures of Cry34Ab1and Cry35Ab1 were observed as out-group. In case ofDiptera, 3-dimensional structures of Cry4Ba were founddiverged from other Cry proteins whereas Cry2Ab proteinof Lepidoptera group noticed to be diverged from othergroup of proteins. However, Cry15aa was predicted as anout-group in compare to other Cry proteins.

ConclusionsBacillus thuringiensis (Bt) synthesizes various insecticidalproteins and thus recommended as potential bio controlagent against various insect pests in agriculture. Theevolution and diversification of these Cry proteins have

Fig. 7 a 3D Structure alignment result using Phyre2 server of Cry proteins (Coleoptera). b 3D Structure alignment result using Phyre2 server of CryProteins (Diptera). c 3D Structure alignment result using Phyre2 server of Cry Proteins (Lepidoptera)

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been studied extensively by various researchers to dis-cover the existence of important determinants, whichconfer insect specificity for improvement of its insecti-cidal activity. There are total 58 numbers of differentCry protein groups belong to major three insect orders:Coleoptera, Diptera, and Lepidoptera were retrieved andanalyzed both at structural and sequence level. Struc-tural and functional analysis was performed to under-stand the domain arrangements at sequence andstructural level involving both experimental and pre-dicted 3D models. Cry proteins having toxicity for a spe-cific insect order are grouped accordingly. Three-dimensional structure analysis of both experimental andpredicted models revealed that the Cry proteins mighthave toxicity for a specific insect order differ in theirstructural arrangements and is observed in 3 differentgroups. It could be hypothesized that an inner-moleculardomain shift or domain insertion/deletion might havetaken place during the evolutionary process, which con-sequently causes structural and functional divergence ofBt. These findings lead to understand the wide diversityof insecticidal proteins and their application as biopesti-cides in agriculture.

AbbreviationsBt: Bacillus thuringiensis; PFTs: Pore-forming toxins; 3D: Three-dimensional;CADs: Cadherin-like proteins; APN: Aminopeptidase N; UniProt: UniversalProtein Resource; NCBI : National Center for Biotechnology Information; CD-Search: Conserved Domain-Search; RCSB-PDB: Research Collaboratory forStructural Bioinformatics-Protein Data Bank; MEGA X: Molecular EvolutionaryGenetics Analysis X; Phyre2: Protein Homology/analogY Recognition Engine V2.0

AcknowledgementsFinancial assistance to the Center of Excellence in Environment and PublicHealth by the Higher Education Department, Government of Odisha underOHEPEE is grateful acknowledged (HE-PTC-WB-02017).

Authors’ contributionsSKD: Research concept and design, collection and/or assembly of data, dataanalysis and interpretation, writing the article, and critical revision of thearticle. SKP: Research concept and design, and data analysis andinterpretation. KCS: Data analysis and interpretation, and critical revision ofthe article. NRS: Research concept and design. The authors read andapproved the final manuscript.

FundingNot applicable.

Availability of data and materialsThe datasets used and/or analyzed during the current study are available inthe UniProtKB (The Universal Protein Resource Knowledgebase).

Ethics approval and consent to participateNot applicable.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Department of Bioinformatics, Centre for Post Graduate Studies, OdishaUniversity of Agriculture & Technology, Bhubaneswar, Odisha 751003, India.2School of Life Sciences, Ravenshaw University, Cuttack, Odisha 753003, India.3Department of Agricultural Biotechnology, College of Agriculture, OdishaUniversity of Agriculture & Technology, Bhubaneswar, Odisha 751003, India.4Department of Botany and Center of Excellence in Environment and PublicHealth, Ravenshaw University, Cuttack, Odisha 753003, India.

Received: 27 August 2020 Accepted: 17 February 2021

ReferencesBayyareddy K, Andacht TM, Abdullah MA, Adang MJ (2009) Proteomic

identification of Bacillus thuringiensis subsp. israelensis toxin Cry4Ba bindingproteins in midgut membranes from Aedes aegyptiLinnaeus (Diptera,Culicidae) larvae. Insect Biochem Mol Biol 39(4):279–286. https://doi.org/10.1016/j.ibmb.2009.01.002

Boonserm P, Davis P, Ellar DJ, Li J (2005) Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications. J Mol Biol 348(2):363–382. https://doi.org/10.1016/j.jmb.2005.02.013

Boonserm P, Mo M, Angsuthanasombat C, Lescar J (2006) Structure of thefunctional form of the mosquito larvicidal Cry4Aa toxin from Bacillusthuringiensis at a 2.8-angstrom resolution. J Bacteriol 188(9):3391–3401.https://doi.org/10.1128/JB.188.9.3391-3401.2006

Bravo A, Gill SS, Soberón M (2007) Mode of action of Bacillus thuringiensis Cryand Cyt toxins and their potential for insect control. Toxicon 49(4):423–435.https://doi.org/10.1016/j.toxicon.2006.11.022

Bravo A, Gómez I, Porta H, García-Gómez BI, Rodrigue-Almazan C, Pardo L,Soberón M (2013) Evolution of Bacillus thuringiensis Cry toxins insecticidalactivity. J Microbial Biotechnol 6(1):17–26. https://doi.org/10.1111/j.1751-7915.2012.00342.x

Bravo A, Likitvivatanavong S, Gill SS, Soberón M (2011) Bacillus thuringiensis: astory of a successful bioinsecticide. Insect Biochem Mol Biol 41(7):423–431.https://doi.org/10.1016/j.ibmb.2011.02.006

Choma CT, Kaplan H (1990) Folding and unfolding of the protoxin from Bacillusthuringiensis: evidence that the toxic moiety is present in an activeconformation. Biochemistry 29(49):10971–10977. https://doi.org/10.1021/bi00501a015

Crickmore N (2000) The diversity of Bacillus thuringiensis δ-endotoxins. In:Entomopathogenic bacteria: from laboratory to field application. Springer,Dordrecht, pp 65–79

Crickmore N, Zeigler DR, Feitelson J, Schnepf ES, Van Rie J, Lereclus D, Baum J,Dean DH (1998) Revision of the nomenclature for the Bacillus thuringiensispesticidal crystal proteins. Microbiol Mol Biol Rev 62(3):807–813. https://doi.org/10.1128/MMBR.62.3.807-813.1998

Crickmore N, Zeigler DR, Schnepf E, Van Rie J, Lereclus D, Baum J (2011)Bacillusthuringiensis toxin. [WWWdocument].URL http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/.

de Maagd RA, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evolvedspecific toxins to colonize the insect world. Trends Genet 17(4):193–199.https://doi.org/10.1016/S0168-9525(01)02237-5

Derbyshire DJ, Ellar DJ, Li J (2001) Crystallization of the Bacillus thuringiensis toxinCry1Ac and its complex with the receptor ligand N-acetyl-D-galactosamine.Acta Crystallogr D Biol Crystallogr 57(12):1938–1944. https://doi.org/10.1107/S090744490101040X

Donovan WP, Engleman JT, Donovan JC, Baum JA, Bunkers GJ, Chi DJ, ClintonWP, English L, Heck GR, Ilagan OM, Krasomil-Osterfeld KC (2016) Discoveryand characterization of Sip1A: a novel secreted protein from Bacillusthuringiensis with activity against coleopteran larvae. Appl MicrobiolBiotechnol 72(4):713–719. https://doi.org/10.1007/s00253-006-0332-7

Evdokimov AG, Moshiri F, Sturman EJ, Rydel TJ, Zheng M, Seale JW, Franklin S(2014) Structure of the full length insecticidal protein Cry1 A c revealsintriguing details of toxin packaging into in vivo formed crystals. Protein Sci23(11):1491–1497. https://doi.org/10.1002/pro.2536

Galitsky N, Cody V, Wojtczak A, Ghosh D, Luft JR, Pangborn W, English L (2001)Structure of the insecticidal bacterial δ-endotoxin Cry3Bb1 of Bacillusthuringiensis. Acta Crystallogr D Biol Crystallogr 57(8):1101–1109. https://doi.org/10.1107/S0907444901008186

Das et al. Egyptian Journal of Biological Pest Control (2021) 31:44 Page 13 of 14

Gouet P, Robert X, Courcelle E (2003) ESPript/ENDscript: extracting and renderingsequence and 3D information from atomic structures of proteins. NucleicAcids Res 31(13):3320–3323. https://doi.org/10.1093/nar/gkg556

Griffitts JS, Haslam SM, Yang T, Garczynski SF, Mulloy B, Morris H, Cremer PS, DellA, Adang MJ, Aroian RV (2005) Glycolipids as receptors for Bacillusthuringiensis crystal toxin. Science 307(5711):922–925. https://doi.org/10.1126/science.1104444

Grochulski P, Masson L, Borisova S, Pusztai-Carey M, Schwartz JL, Brousseau R,Cygler M (1995) Bacillus thuringiensis CrylA (a) insecticidal toxin: crystalstructure and channel formation. J Mol Biol 254(3):447–464. https://doi.org/10.1006/jmbi.1995.0630

Guo S, Ye S, Liu Y, Wei L, Xue J, Wu H, Song F, Zhang J, Wu X, Huang D, Rao Z(2009) Crystal structure of Bacillus thuringiensis Cry8Ea1: an insecticidal toxintoxic to underground pests, the larvae of Holotrichia parallela. J Struct Biol168(2):259–266. https://doi.org/10.1016/j.jsb.2009.07.004

Höfte H, Whiteley HR (1989) Insecticidal crystal proteins of Bacillus thuringiensis.Microbiol Mol Biol Rev 53(2):242–255

Hui F, Scheib U, Hu Y, Sommer RJ, Aroian RV, Ghosh P (2012) Structure andglycolipid binding properties of the nematicidal protein Cry5B. Biochemistry51(49):9911–9921. https://doi.org/10.1021/bi301386q

Kelker MS, Berry C, Evans SL, Pai R, McCaskill DG, Wang NX, Russell JC, Baker MD,Yang C, Pflugrath JW, Wade M (2014) Structural and biophysical characterizationof Bacillus thuringiensis insecticidal proteins Cry34Ab1 and Cry35Ab1. PLoS One9(11):e112555. https://doi.org/10.1371/journal.pone.0112555

Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecularevolutionary genetics analysis across computing platforms. Mol Biol Evol35(6):1547–1549. https://doi.org/10.1093/molbev/msy096

Li J, Carroll J, Ellar DJ (1991) Crystal structure of insecticidal δ-endotoxin fromBacillus thuringiensisat 2.5 Å resolution. Nature 353(6347):815–821. https://doi.org/10.1038/353815a0

López-Pazos SA, Gómez JE, Salamanca JA (2009) Cry1B and Cry3A are activeagainst Hypothenemus hampei Ferrari (coleoptera: scolytidae). J InvertebrPathol 101(3):242–245

McNall RJ, Adang MJ (2003) Identification of novel Bacillus thuringiensis Cry1Acbinding proteins in Manduca sexta midgut through proteomic analysis.Insect Biochem Mol Biol 33(10):999–1010. https://doi.org/10.1016/S0965-1748(03)00114-0

Mizuki E, Park YS, Saitoh H, Yamashita S, Akao T, Higuchi K, Ohba M (2000)Parasporin, a human leukemic cell-recognizing parasporal protein of Bacillusthuringiensis. Clin Diagn Lab Immunol 7(4):625–634. https://doi.org/10.1128/CDLI.7.4.625-634.2000

Morse RJ, Yamamoto T, Stroud RM (2001) Structure of Cry2Aa suggests anunexpected receptor binding epitope. Structure 9(5):409–417. https://doi.org/10.1016/S0969-2126(01)00601-3

Naimov S, Boncheva R, Karlova R, Dukiandjiev S, Minkov I, de Maagd RA (2008)Solubilization, activation, and insecticidal activity of Bacillus thuringiensisserovarthompsoni HD542 crystal proteins. Appl Environ Microbiol 74(23):7145–7151. https://doi.org/10.1128/AEM.00752-08

Palma L, Muñoz D, Berry C, Murillo J, Caballero P (2014) Bacillus thuringiensistoxins: an overview of their biocidal activity. Toxins 6(12):3296–3325. https://doi.org/10.3390/toxins6123296

Pigott CR, Ellar DJ (2007) Role of receptors in Bacillus thuringiensis crystal toxinactivity. Microbiol Mol Biol Rev 71(2):255–281. https://doi.org/10.1128/MMBR.00034-06

Raymond B, Johnston PR, Nielsen-LeRoux C, Lereclus D, Crickmore N (2010)Bacillus thuringiensis: an impotent pathogen? Trends Microbiol 18(5):189–194.https://doi.org/10.1016/j.tim.2010.02.006

Saitou N, Nei M (1987) The neighbor-joining method: a new method forreconstructing phylogenetic trees. Molecular biology and evolution 4(4):406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454

Sanchis V, Bourguet D (2008) Bacillus thuringiensis: applications in agriculture andinsect resistance management. A review. Agronom Sustain Devs 28(1):11–20.https://doi.org/10.1051/agro:2007054

Schnepf E, Crickmore NV, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR,Dean D (1998) Bacillus thuringiensis and its pesticidal crystal proteins.Microbiol Mol Biol Rev 62(3):775–806. https://doi.org/10.1128/MMBR.62.3.775-806.1998

Schnepf HE (1995) Bacillus thuringiensis toxins: regulation, activities and structuraldiversity. Curr Opin Biotechnol 6(3):305–312. https://doi.org/10.1016/0958-1669(95)80052-2

Soberon M, Gill SS, Bravo A (2009) Signaling versus punching hole: How doBacillus thuringiensis toxins kill insect midgut cells? Cell Mol Life Sci 66(8):1337–1349. https://doi.org/10.1007/s00018-00-8330-9

Van Frankenhuyzen K (2009) Insecticidal activity of Bacillus thuringiensis crystalproteins. J Invertebr Pathol 101(1):1–6. https://doi.org/10.1016/j.jip.2009.02.009

Wang C, Li W, Kessenich CR, Petrick JS, Rydel TJ, Sturman EJ, Lee TC, Glenn KC,Edrington TC (2018) Safety of the Bacillus thuringiensis-derived Cry1A. 105protein: evidence that domain exchange preserves mode of action andsafety. Regul Toxicol Pharmacol 99:50–60. https://doi.org/10.1016/j.yrtph.2018.09.003

Wang Y, Wang J, Fu X, Nageotte JR, Silverman J, Bretsnyder EC, Chen D, Rydel TJ,Bean GJ, Li KS, Kraft E (2019) Novel receptor interaction of Bacillusthuringiensis Cry1Da_7 and Cry1B. 868 proteins allow control of resistant fallarmyworm, Spodoptera frugiperda (JE Smith). Appl Environ Microbiol AEM-00579. https://doi.org/10.1128/AEM.00579-19

Zuckerkandl E, Pauling L (1965) Evolutionary divergence and convergence inproteins. Evolving Genes Proteins 1965:97–166. https://doi.org/10.1016/B978-1-4832-2734-4.50017-6

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